Radish cultivar ADS-10

ABSTRACT

A radish cultivar, designated ADS-10, is disclosed. The invention relates to the seeds of radish cultivar ADS-10, to the plants of radish cultivar ADS-10 and to methods for producing a radish plant by crossing radish cultivar ADS-10 with itself or another radish cultivar. The invention further relates to methods for producing a radish plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other radish cultivars derived from radish cultivar ADS-10.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive radish cultivar(Raphanus sativus sp), designated ADS-10. All publications cited hereinare incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include rounder shape, smoother texture, rootsize, higher seed yield, improved color, resistance to diseases andinsects, tolerance to drought and heat, and better agronomic quality.

All cultivated forms of radish belong to the Family Cruciferae (alt.Brassicaceae), and are grown for their edible hypocotyl. Radishes havebeen cultivated for thousands of years in both China and theMediterranean areas. Generally, commercial radishes are grown whereverenvironmental conditions permit the production of an economically viableyield. The radish grown in the United States is primarily an annual,although biennial types occur. In the United States, the top producingstates for radish (Raphanus sativus) are Florida (32 percent),California (20 percent), Michigan (16 percent), Minnesota (10 percent),and Ohio (7 percent). Fresh radish is available in the United Statesyear-round, where domestic supplies are the highest from May to October,while imports are at their peak from November to April. For plantingpurposes, radishes grow best in rather cool weather—fall and spring ofthe Northern states and late fall, winter, and early spring in thewarmer states. Radish is consumed mainly as a salad plant and eaten raw,but can be eaten as a cooked or pickled vegetable.

Radish is a quick growing, primarily annual, cool season root vegetablethat matures in 3 to 6 weeks. The seed will germinate in 3 to 4 dayswith soil temperatures of 18° C. to 30° C., but germination ratesdecline sharply when the soil temperatures fall below 13° C. The bestquality and root shape are obtained when the crop grows and matures atmoderate temperatures of 10° C. to 30° C. in intermediate to short daylengths. When grown in hot weather, radish tend to elongate, developpoor shape or no edible hypocotyl at all, and become more pungent. Whengrown in cold weather, radish tops grow larger and taller, while longdays induce flowering or bolting. Thus, growth must be continuous andrapid for good quality. Radish remain in prime condition only for a fewdays, as the edible hypocotyl remains in marketable condition only ashort time before becoming pithy.

A study of cross-pollination of the radish indicated that the varieties‘Icicle’ and ‘Scarlet Globe’ were self-incompatible and that crossingdecreased from 30 to 40 percent when these two varieties were placed at9 inches apart to 0.1 percent when at 240 feet apart. Crane, M. B., andMather, K. Ann. Appl. Biol. 30: 301-308. 1943. Another study indicatedthat honey bees are the most important agents in the pollination of theradish, enhancing the seed crop and increasing crop yield by 22 percent.Radchenko, T. H. Mich. Agr. Expt. Sta. Quart. Bul. 27: 413-420. 1966.These and other studies appear to agree that the radish is almostentirely insect-pollinated.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, pure linecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of radish plant breeding is to develop new, unique and superiorradish cultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations. The breeder has no direct control at the cellular level.Therefore, two breeders will never develop the same line, or even verysimilar lines, having the same radish traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior radish cultivars.

The development of new commercial radish cultivars requires thedevelopment of radish varieties, the crossing of these varieties, andthe evaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits, are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population, will be represented by a progenywhen generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen, (Molecular Linkage Map ofSoybean (Glycine max) p 6.131-6.138 in S. J. O'Brien (ed) Genetic Maps:Locus Maps of Complex Genomes, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1993)) developed a molecular genetic linkage mapthat consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, threeclassical markers and four isozyme loci. See also, Shoemaker, R. C.,RFLP Map of Soybean, p 299-309, in Phillips, R. L. and Vasil, I. K.,eds. DNA-Based Markers in Plants, Kluwer Academic Press, Dordrecht, theNetherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. (Diwan, N. and Cregan, P.B., Theor. Appl. Genet. 95:22-225,1997.) SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intoradish varieties. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation (such as X-rays, Gamma rays, neutrons,Beta radiation, or ultraviolet radiation), chemical mutagens (such asbase analogs like 5-bromo-uracil), antibiotics, alkylating agents (suchas sulfur mustards, nitrogen mustards, epoxides, ethyleneamines,sulfates, sulfonates, sulfones, or lactones), azide, hydroxylamine,nitrous acid or acridines. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in “Principles of Cultivar Development” by Fehr, MacmillanPublishing Company, 1993.

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan et al., Theor. Appl. Genet., 77:889-892, 1989.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Radish in general is an important and valuable vegetable crop. Thus, acontinuing goal of plant breeders is to develop stable, high yieldingradish cultivars that are agronomically sound. The reasons for this goalare obviously to maximize the amount of yield produced on the land. Toaccomplish this goal, the radish breeder must select and develop radishplants that have the traits that result in superior cultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel radish cultivar,designated ADS-10. This invention thus relates to the seeds of radishcultivar ADS-10, to the plants of radish cultivar ADS-10, and to methodsfor producing a radish plant produced by crossing the radish ADS-10 withitself or another radish cultivar, to methods for producing a radishplant containing in its genetic material one or more transgenes and tothe transgenic radish plants produced by that method, and the creationof variants by mutagenesis or transformation of radish cultivar ADS-10.This invention also relates to methods for producing other radishcultivars derived from radish cultivar ADS-10 and to the radish cultivarderived by the use of those methods. This invention further relates tohybrid radish seeds and plants produced by crossing the cultivar ADS-10with another radish cultivar.

Thus, any such methods using the radish cultivarADS-10 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar ADS-10 asat least one parent are within the scope of this invention.Advantageously, the radish cultivar could be used in crosses with other,different, radish plants to produce first generation (F₁) radish hybridseeds and plants with superior characteristics.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of radish cultivar ADS-10. The tissue culture willpreferably be capable of regenerating plants having all of thephysiological and morphological characteristics of the foregoing radishplant, and of regenerating plants having substantially the same genotypeas the foregoing radish plant. Preferably, the regenerable cells in suchtissue cultures will be embryos, protoplasts, seeds, hypocotyls, pollen,leaves, pistils, anthers, flowers, roots, root tips, stems, andmeristematic cells. Still further, the present invention provides radishplants regenerated from the tissue cultures of the invention.

Another aspect of the invention is to provide methods for producingother radish plants derived from radish cultivar ADS-10. Radishcultivars derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a radish plantcontaining in its genetic material one or more transgenes and to thetransgenic radish plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of ADS-10. The single transferred gene may preferablybe a dominant or recessive allele. Preferably, the single transferredgene will confer such trait as male sterility, herbicide resistance,insect resistance, resistance for bacterial, fungal, or viral disease,male fertility, enhanced nutritional quality and industrial usage. Thesingle gene may be a naturally occurring radish gene or a transgeneintroduced through genetic engineering techniques.

The invention further provides methods for developing a radish plant ina radish plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection and transformation. Seeds, radish plant, andparts thereof produced by such breeding methods are also part of theinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which alleles relates to one trait or characteristic. In adiploid cell or organism, the two alleles of a given gene occupycorresponding loci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all of the phvsiological and morphological characteristics.A plant having essentially all of the physiological and morphologicalcharacteristics means a plant having all of the physiological andmorphological characteristics, except for the characteristics derivedfrom the converted gene.

Maturity Date. Maturity in radish can be dictated by two conditions. Thefirst, or market maturity, is the point in time when the radish reachesmaximum size distribution, but before defects such as pith appear. Thesecond, or seed maturity, is the biological maturity when the plant hascompleted the life cycle and produced viable seed.

MUN. MUN refers to the MUNSELL Color Chart which publishes an officialcolor chart for plant tissues according to a defined numbering system.The chart may be purchased from the Macbeth Division of KollmorgenInstruments Corporation; 617 Little Britain Road; New Windsor, N.Y.,12553-6148.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a line are recovered in addition to thesingle gene transferred into the line via the backcrossing technique orvia genetic engineering.

Radish Yield (Boxes/Acre). The yield in boxes/acre is the actual yieldof the radish at harvest.

Pith. Pith is a sponginess/hollowness/white discoloration that occurs inthe hypocotyl of radish varieties naturally as they become over mature.In some varieties, it occurs at an earlier stage causing harvest tooccur prior to ideal maturity.

DETAILED DESCRIPTION OF THE INVENTION

Radish cultivar ADS-10 is a red globe, fresh market radish. A unique andimportant trait of this variety is that the hypocotyl holds the roundshape under a wide variety of environmental conditions. This round shapeis even maintained under adverse weather conditions making it truly aunique variety. Another important characteristic of the presentinvention is that the shoulder of the radish stays smooth without anydevelopment of scurf or hypocotyl scarring. The root attachment is veryabrupt with typically a 3 mm diameter at the point of attachment. Theinterior is white but some red coloration may develop under adverseconditions. An important trait of this cultivar is that it is an openpollinated variety.

Some of the criteria used for selection in various generations include:color, number of leaves, shape, yield, emergence, maturity, plantarchitecture, seed yield and quality, and disease resistance.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. The line has beenincreased with continued observation for uniformity. No variant traitshave been observed or are expected in ADS-10.

Radish cultivar ADS-10 has the following morphologic and othercharacteristics (based primarily on data collected at Belle Glade,Fla.). The colors of the various plant parts are detailed with referenceto the Munsell Color Reference Chart. TABLE 1 VARIETY DESCRIPTIONINFORMATION Plant Characteristics: Days from sowing to market maturity:31 Number of internodes: 12 Plant height at seed maturity: 108 cm Leaf:Length: 21 cm Width: 5.4 cm Number: 6 true leaves Pairs of lateralpinnae: 4 Texture: Hirsute Color: MUN 5 GY 5/6 (dark green) Flowers:Color: Pink at the tips and white in the center Number of flowers perraceme: 41 Vein color: Non-distinct Silique: Length: 5.2 cm Maximumdiameter: 12 mm Medium number of seeds per silique: 5 Seed color:Red-brown Hypocotyl: Shape: Round L/D ratio: 1.0 Color: MUN 5 R 4/8(red) Skin texture: Smooth Taproot size: Fine

This invention also is directed to methods for producing a radish plantby crossing a first parent radish plant with a second parent radishplant wherein either the first or second parent radish plant is a radishplant of radish cultivar ADS-10. Further, both first and second parentradish plants can come from the cultivar ADS-10. Still further, thisinvention also is directed to methods for producing a cultivarADS-10-derived radish plant by crossing cultivar ADS-10 with a secondradish plant and growing the progeny seed, and repeating the crossingand growing steps with the cultivar ADS-10-derived plant from 0 to 7times. Thus, any such methods using the cultivar ADS-10 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar ADS-10 asa parent are within the scope of this invention, including plantsderived from cultivar ADS-10. Advantageously, cultivar ADS-10 can beused in crosses with other, different, cultivars to produce firstgeneration (F₁) radish seeds and plants with superior characteristics.

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably, expression vectors are introduced into planttissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

TABLES

In the tables that follow, the traits and characteristics of radishcultivar ADS-10 are given compared to a standard radish variety, RedSilk.

Yield in radish is highly dependent on the size of the radish atharvest, which is further dependent on the growing time in the field.The longer the radish is allowed to grow in the field, the higher theyield. However, for commercial purposes, it is not desirable to allowthe radish to reach maximum size.

Table 2 shows the yield in boxes per acre of ADS-10 compared to thestandard variety, Red Silk, in Belle Glade, Fla. The yield of ADS-10 islisted solely to demonstrate that it is comparable to average yield ofRed Silk. TABLE 2 Harvest Date ADS-10 (boxes/acre) Red Silk (boxes/acre)Dec. 16, 2002 470 550 Mar. 04, 2003 643 550 Mar. 21, 2003 683 550

A rounder shape is one of the main attributes of this variety that makesit desirable for use as a commercial market radish. Table 3 shows thepercentage of radishes with an L/D ratio greater than 1.2 underdifferent environmental conditions in Belle Glade, Fla., as shown by thevarious harvest dates. TABLE 3 Evaluation Date ADS-10 Red Silk Nov. 21,2001 0 15 Nov. 30, 2001 0 70 Jan. 03, 2002 0 45 Nov. 09, 2002 0 80 Dec.13, 2002 15 30 Feb. 03, 2003 0 30 Apr. 19, 2003 30 60 Feb. 23, 2004 0 3

A more abrupt root attachment is another main attribute of this variety.Table 4 shows the percentage of swollen hypocotyls with a root diameterat the point of attachment less than 12% of the total hypocotyl'sdiameter measured at the widest point. TABLE 4 Evaluation Date ADS-10Red Silk Nov. 21, 2001 85 35 Nov. 30, 2001 70 10 Jan. 03, 2002 70 35Nov. 09, 2002 100 35 Dec. 13, 2002 45 25 Feb. 03, 2003 95 80 Apr. 19,2003 65 55 Feb. 23, 2004 90 54

FURTHER EMBODIMENTS OF THE INVENTION

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed radish plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the radish plant(s).

Expression Vectors for Radish Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalswhich confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad.Sci. U.S.A., 80:4803 (1983). Another commonly used selectable markergene is the hygromycin phosphotransferase gene which confers resistanceto the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol.,5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990) Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or bromoxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988). Selectable marker genes forplant transformation not of bacterial origin include, for example, mousedihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphatesynthase and plant acetolactate synthase. Eichholtz et al., Somatic CellMol. Genet 13:67 (1987), Shah et al., Science 233:478 (1986), Charest etal., Plant Cell Rep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include α-glucuronidase (GUS, α-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, IMAGENE GREEN, p.1-4 (1993) and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Radish Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression inradish. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in radish. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); ln2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression inradish or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in radish.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)). The ALS promoter, Xba1/NcoI fragment 5′ to the Brassicanapus ALS3 structural gene (or a nucleotide sequence similarity to saidXba1/NcoI fragment), represents a particularly useful constitutivepromoter. See PCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin radish. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in radish. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targetinq Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is radish. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

-   1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest: For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin CDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin CDNA clone.

L. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa celery endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoradish in order to increase its resistance to LMV infection. See Dinantet al., Molecular Breeding. 1997, 3:1, 75-86.

-   2. Genes That Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvishikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy proprionic acids and cyclohexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses Lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,Degreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy proprionic acids and cyclohexones, suchas sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chiamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori et al., Mol. Gen. Genet246:419, 1995. Other genes that confer tolerance to herbicides include agene encoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase (Shiota et al., Plant Physiol.,106:17, 1994), genes for glutathione reductase and superoxide dismutase(Aono et al., Plant Cell Physiol. 36:1687, 1995), and genes for variousphosphotransferases (Datta et al., Plant Mol. Biol. 20:619, 1992).

E. Protoporphyrinogen oxidase (protox) is necessaryforthe production ofchlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837;5,767,373; and international publication WO 01/12825.

-   3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the radish, for example by transforming aplant with a soybean ferritin gene as described in Goto et al., ActaHorticulturae. 2000, 521, 101-109.

B. Decreased nitrate content of leaves, for example by transforming aradish with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999,18: 11, 889-896.

C. Increased sweetness of the radish by transferring a gene coding formonellin, that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), S{acute over (o/)}gaard et al., J. Biol. Chem. 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene), and Fisher et al.,Plant Physiol. 102:1045 (1993) (maize endosperm starch branching enzymeII).

-   4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the bamase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622,1992).

Methods for Radish Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

-   A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

-   B. Direct Gene Transfer

Several methods of plant transformation, collectively referred to asdirect gene transfer, have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 to 4 μm.The expression vector is introduced into plant tissues with a biolisticdevice that accelerates the microprojectiles to speeds of 300 to 600 m/swhich is sufficient to penetrate plant cell walls and membranes.Russell, D. R., et al. Pl. Cell. Rep. 12(3, Jan.), 165-169 (1993),Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, Oct.), 357-359 (1992),Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490 (1993).Aragao Theor. Appl. Genet. 93:142-150 (1996), Kim, J.; Minamikawa, T.Plant Science 117: 131-138 (1996), Sanford et al., Part. Sci. Technol.5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol. Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992)

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of radish target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic radish cultivar. Alternatively, a genetic trait whichhas been engineered into a particular radish cultivar using theforegoing transformation techniques could be moved into another cultivarusing traditional backcrossing techniques that are well known in theplant breeding arts. For example, a backcrossing approach could be usedto move an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Single-Gene Conversions

When the term radish plant, cultivar or radish line are used in thecontext of the present invention, this also includes any single geneconversions of that cultivar. The term “single gene converted plant” asused herein refers to those radish plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into thecultivar via the backcrossing technique. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the cultivar. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to one of the parental radishplants for that cultivar, backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or moretimes to the recurrent parent. The parental radish plant whichcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental radish plant to which the gene orgenes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcrossprotocol, the original cultivar of interest (recurrent parent) iscrossed to a second line (nonrecurrent parent) that carries the singlegene of interest to be transferred. The resulting progeny from thiscross are then crossed again to the recurrent parent and the process isrepeated until a radish plant is obtained wherein essentially all of thedesired morphological and physiological characteristics of the recurrentparent are recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent, as determined at the 5%significance level when grown in the same environmental conditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalcultivar. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new cultivar but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic, examples of these traits include but are not limited to,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, enhanced nutritional quality, industrialusage, yield stability and yield enhancement. These genes are generallyinherited through the nucleus. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196, 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

Tissue Culture

As is well known in the art, tissue culture of radish can be used forthe in vitro regeneration of a radish plant. Tissue culture of varioustissues of plants and regeneration of plants therefrom is well known andwidely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9,1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000,125: 6, 669-672. Thus, another aspect ofthis invention is to provide cells which upon growth and differentiationproduce radish plants having all of the physiological and morphologicalcharacteristics of radish variety ADS-10.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, hypocotyls, pollen, flowers, seeds,leaves, stems, roots, root tips, anthers, and the like. Means forpreparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

This invention also is directed to methods for producing a radish plantby crossing a first parent radish plant with a second parent radishplant wherein the first or second parent radish plant is a radish plantof cultivar ADS-10. Further, both first and second parent radish plantscan come from radish cultivar ADS-10. Thus, any such methods usingradish cultivar ADS-10 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using radish cultivar ADS-10 as at least one parent are withinthe scope of this invention, including those developed from cultivarsderived from radish cultivar ADS-10. Advantageously, this radishcultivar could be used in crosses with other, different, radish plantsto produce the first generation (F₁) radish hybrid seeds and plants withsuperior characteristics. The cultivar of the invention can also be usedfor transformation where exogenous genes are introduced and expressed bythe cultivar of the invention. Genetic variants created either throughtraditional breeding methods using radish cultivar ADS-10 or throughtransformation of cultivar ADS-10 by any of a number of protocols knownto those of skill in the art are intended to be within the scope of thisinvention.

The following describes breeding methods that may be used with radishcultivar ADS-10 in the development of further radish plants. One suchembodiment is a method for developing a progeny radish plant in a radishplant breeding program comprising: obtaining the radish plant, or a partthereof, of cultivar ADS-10, utilizing said plant or plant part as asource of breeding material, and selecting a radish cultivar ADS-10progeny plant with molecular markers in common with cultivar ADS-10and/or with morphological and/or physiological characteristics selectedfrom the characteristics listed in Tables 1, 2, 3 or 4. Breeding stepsthat may be used in the radish plant breeding program include pedigreebreeding, backcrossing, mutation breeding, and recurrent selection. Inconjunction with these steps, techniques such as RFLP-enhancedselection, genetic marker enhanced selection (for example SSR markers)and the making of double haploids may be utilized.

Another method involves producing a population of radish cultivar ADS-10progeny radish plants, comprising crossing cultivar ADS-10 with anotherradish plant, thereby producing a population of radish plants, which, onaverage, derive 50% of their alleles from radish cultivar ADS-10. Aplant of this population may be selected and repeatedly selfed or sibbedwith a radish cultivar resulting from these successive filialgenerations. One embodiment of this invention is the radish cultivarproduced by this method and that has obtained at least 50% of itsalleles from radish cultivar ADS-10.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, p 261-286 (1987). Thus the invention includes radishcultivar ADS-10 progeny radish plants comprising a combination of atleast two cultivarADS-10 traits selected from the group consisting ofthose listed in Tables 1, 2, 3 or 4 or the cultivar ADS-10 combinationof traits listed in the Summary of the Invention, so that said progenyradish plant is not significantly different for said traits than radishcultivar ADS-10 as determined at the 5% significance level when grown inthe same environmental conditions. Using techniques described herein,molecular markers may be used to identify said progeny plant as a radishcultivar ADS-10 progeny plant. Mean trait values may be used todetermine whether trait differences are significant, and preferably thetraits are measured on plants grown under the same environmentalconditions. Once such a variety is developed its value is substantialsince it is important to advance the germplasm base as a whole in orderto maintain or improve traits such as yield, disease resistance, pestresistance, and plant performance in extreme environmental conditions.

Progeny of radish cultivar ADS-10 may also be characterized throughtheir filial relationship with radish cultivar ADS-10, as for example,being within a certain number of breeding crosses of radish cultivarADS-10. A breeding cross is a cross made to introduce new genetics intothe progeny, and is distinguished from a cross, such as a self or a sibcross, made to select among existing genetic alleles. The lower thenumber of breeding crosses in the pedigree, the closer the relationshipbetween radish cultivar ADS-10 and its progeny. For example, progenyproduced by the methods described herein may be within 1, 2, 3, 4 or 5breeding crosses of radish cultivar ADS-10.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which radish plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,hypocotyls, roots, root tips, anthers, pistils, flowers, seeds, stems,and the like.

Deposit Information

A deposit of the radish cultivar seed of this invention is maintained byA. Duda & Sons, Inc. 1260 Growers Street, Salinas, Calif. 93902, USA.Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto under 37 CRF 1.14 and 35 USC 122. Uponallowance of any claims in this application, all restrictions on theavailability to the public of the variety will be irrevocably removed byaffording access to a deposit of at least 2,500 seeds of the samevariety with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110 or National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive,Aberdeen, Scotland, AB24 3RY, United Kingdom.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

1. A seed of radish cultivar designated ADS-10, wherein a representativesample of seed of said cultivar was deposited under ATCC Accession No.PTA-______.
 2. A radish plant, or a part thereof, produced by growingthe seed of claim
 1. 3. A tissue culture of cells produced from theplant of claim
 2. 4. A protoplast produced from the tissue culture ofclaim
 3. 5. The tissue culture of claim 3, wherein said cells of thetissue culture are from a plant part selected from the group consistingof leaves, pollen, embryos, hypocotyls, roots, root tips, anthers,pistils, flowers, seeds, and stems.
 6. A radish plant regenerated fromthe tissue culture of claim 3, said radish plant having all of themorphological and physiological characteristics of radish cultivarADS-10, wherein a representative sample of seed of said cultivar wasdeposited under ATCC Accession No. PTA-______.
 7. A method for producingan F₁ hybrid radish seed, comprising crossing the plant of claim 2 witha different radish plant and harvesting the resultant F₁ hybrid radishseed.
 8. A hybrid radish seed produced by the method of claim
 7. 9. Ahybrid radish plant, or a part thereof, produced by growing said hybridseed of claim
 8. 10. A method for producing a male sterile radish plantcomprising transforming the radish plant of claim 2 with a nucleic acidmolecule that confers male sterility.
 11. A male sterile radish plantproduced by the method of claim
 10. 12. A method of producing anherbicide resistant radish plant comprising transforming the radishplant of claim 2 with a transgene that confers herbicide resistance. 13.An herbicide resistant radish plant produced by the method of claim 12.14. The radish plant of claim 13, wherein the transgene confersresistance to an herbicide selected from the group consisting ofimidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine, and benzonitrile.
 15. A method ofproducing an insect resistant radish plant comprising transforming theradish plant of claim 2 with a transgene that confers insect resistance.16. An insect resistant radish plant produced by the method of claim 15.17. The radish plant of claim 16, wherein the transgene encodes aBacillus thuringiensis endotoxin.
 18. A method of producing a diseaseresistant radish plant comprising transforming the radish plant of claim2 with a transgene that confers disease resistance.
 19. A diseaseresistant radish plant produced by the method of claim
 18. 20. A methodof producing a radish plant with modified fatty acid metabolism ormodified carbohydrate metabolism comprising transforming the radishplant of claim 2 with a transgene encoding a protein selected from thegroup consisting of fructosyltransferase, levansucrase, α-amylase,invertase, and starch branching enzyme or encoding an antisense ofstearyl-ACP desaturase.
 21. A radish plant produced by the method ofclaim
 20. 22. A method of introducing a desired trait into radishcultivar ADS-10 comprising: (a) crossing radish cultivar ADS-10 plantsgrown from radish cultivar ADS-10 seed, wherein a representative sampleof seed was deposited under ATCC Accession No. PTA-______, with plantsof another radish cultivar that comprise and express a desired trait toproduce progeny plants, wherein the desired trait is selected from thegroup consisting of male sterility, herbicide resistance, insectresistance and disease resistance; (b) selecting one or more progenyplants that have and express the desired trait to produce selectedprogeny plants; (c) crossing the selected progeny plants with the radishcultivar ADS-10 plants to produce backcross progeny plants; (d)selecting for backcross progeny plants that have and express the desiredtrait and all of the physiological and morphological characteristics ofradish cultivar ADS-10 listed in Table 1 to produce selected backcrossprogeny plants; and (e) repeating steps (c) and (d) three or more timesin succession to produce selected fourth or higher backcross progenyplants that comprise the desired trait and all of the physiological andmorphological characteristics of radish cultivar ADS-10 listed inTable
 1. 23. A radish plant produced by the method of claim 22, whereinthe plant has and expresses the desired trait and all of thephysiological and morphological characteristics of radish line ADS-10listed in Table
 1. 24. The radish plant of claim 23, wherein the desiredtrait is herbicide resistance and the resistance is conferred by atransgene to an herbicide selected from the group consisting ofimidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 25. The radish plant ofclaim 23, wherein the desired trait is insect resistance and the insectresistance is conferred by a transgene encoding a Bacillus thuringiensisendotoxin.
 26. The radish plant of claim 23, wherein the desired traitis male sterility and the trait is conferred by a cytoplasmic nucleicacid molecule that confers male sterility.